Knowledge of the monoamine transporters structural elements that define substrates and inhibitors is sparse. We have recently addressed this structure-activity question directly by generating a series of 3,4-methylenedioxy ring-substituted amphetamine analogs that differ only in the number of methyl substituents on the amine group. Starting with 3,4,-methylenedioxy-N-methylamphetamine (MDMA), 3,4,-methylenedioxy-N,N-dimethylamphetamine (MDDMA) and 3,4,-methylenedioxy-N,N,N-trimethylamphetamine (MDTMA) were prepared. We evaluated functional activities of the analogues at all three monoamine transporters in native brain tissue and in cells expressing transporters, and used ligand docking to generate models of the respective protein-ligand complexes. This approach allowed us to relate experimental findings to available structural information. Our results suggest that 3,4-methylenedioxy amphetamine analogs bind at the orthosteric binding site (OBS) of transporters by adopting one of two mutually exclusive binding modes. MDA and MDMA adopt a high-affinity binding mode, whereas MDDMA and MDTMA adopt a low-affinity binding mode in which the ligand orientation is inverted. Importantly, MDDMA can alternate between both binding modes while MDTMA exclusively binds to the low affinity mode. Our experimental results are consistent with the idea that the initial orientation of bound ligands is critical for subsequent interactions that lead to transporter conformational changes and substrate translocation. The SSRI citalopram (CIT) is a racemic mixture of (+)- and (-) enantiomers, of which the latter (S-(+)-CIT) is the more active antidepressant and currently marketed as Escitalopram. Previously obtained data suggest that the S-(+)-CIT also binds to an allosteric site (S2) that can affect binding at the primary S1 site. To further explore this S2 site, we have designed and synthesized several series of citalopram analogues and separated several sets of enantiomers to further characterize and compare binding profiles at both the SERT and the other monoamine transporters, with the parent ligand. Collectively, these novel SERT compounds are being used to further elucidate structure and function of the SERT by characterizing primary and secondary binding domains that are related in an as of yet undetermined way to behavior. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of fluorescent-derivatives of our tropane based DAT inhibitors. We have identified a high affinity DAT ligand, MFZ 2-12, as an excellent template for a number of useful DAT probes (e.g. 3HMFZ 2-12, 125IMFZ 2-24, JHC 1-064) and have been able to extend our development to Atomic Force Microsopy (AFM) nanoprobes. Using the novel MFZ-AFM tip, an AFM tool was developed to probe the SERT in living cells. By replacing the tropane-based tip with ()-, S- and R-CIT, we relied on nanopharmacological force sensing in physiological conditions, which allowed for the extraction of dynamic information that is inaccessible via X-ray crystallography. Accordingly, our observations unequivocally document the existence of two binding sites on the SERT by tracking binding events to the native membrane at the single molecule level. In addition, the experiments also showed that these two sites are allosterically coupled and exert reciprocal modulation. Our experiments using atomic force spectroscopy provided direct physical evidence of this negative allostery and identified the vestibular S2 binding site as the allosteric site of SERT. Given the importance of the allosteric regulation in biology and pharmacology, our nanopharmacological approach paved a new avenue to explore transient binding sites in clinically-relevant membrane transporters and opened the door to quantitatively address the modulation of interactive forces between ligands and allosterically-coupled binding sites. This project nicely dovetails our S2 probe project. JHC 1-064, has been used to characterize the trafficking and cellular distribution of SERT and DAT in living neuronal cells. Indeed, recent experiments in living neurons with JHC 1-064 have provided data that challenge mechanistic dogma for transporter translocation, as determined in DAT transfected heterologous cells, which is one area of ongoing research with these agents. This fluorescent ligand was recently used in a clinical study to confirm the loss of DAT at the cell membrane in a patient with a genetically mutated DAT that was related to his Parkinsonian and attention deficit comorbidities. Further, JHC 1-064 binds with high affinity to the serotonin transporter (SERT) and the norepinephrine transporter (NET), as well, and we are conducting analogous studies, first in cell lines to study trafficking and cellular distribution of these other monoamine transporters. This project has led to a new project in which we have designed and synthesized novel analogues of the SERT inhibitor and antidepressant, S-citalopram. We have recently prepared a fluorescent citalopram analogue that is currently being used to visualize SERT in living cells.